1. Field of the Invention
The present invention relates to methods and apparatuses for the detection of positional freedom of particles used in biological, biochemical, biophysical, physical, and chemical analyses. Major applications of the present invention include immunodiagnostics, such as blood typing and infectious disease screening.
2. Description of the Related Art
The use of arrayed molecules in formats such as microplates and biochips allows for an ever increasing amount of information to be retrieved about the natural world. Although many variants are known, use of such arrays typically involves immobilizing probes or analytes on a substrate and using a variety of techniques to measure the interaction of the immobilized molecules with other solution-phase molecules. Specific examples of such array techniques include immunoassays (e.g., enzyme-linked immunosorbent assays (ELISAs)) that are performed in microplates, commercial nucleic acid biochips and protein microarrays or biochips. Biochips most commonly use glass substrates, while microplates for ELISA are often constructed from gamma-irradiated polystyrene. Although arrays are typically “ordered”, in the sense that patches of immobilized molecules are placed at defined locations with respect to each other or to reference features, so-called “solution-phase arrays” have also been commercialized. The solution-phase assay technology measures the binding of analytes to a suspension of beads. The suspension is created by mixing batches of particles that are co-labeled with binding probes and corresponding mixtures of two fluorescent identifier molecules in varying ratios. A flow cytometer detects bound fluorescently labeled analyte along with the identifier ratios.
In addition to the detection of individual molecules, particles (including biological cells) have been analyzed using array techniques. For example, it is known in the art to detect cell surface antigens through ELISA or flow cytometry. U.S. Pat. No. 6,251,615 to Oberhardt discloses testing cells using microscopy to detect cells bound to one of a plurality of capture surface regions with coupled antibody receptors.
However, a major limitation of conventional arrays is that long incubation times are usually required to reach a degree of binding that is sufficiently close to equilibrium to achieve the desired sensitivity. This may entail allowing samples or reagents to incubate in an ELISA microplate or biochip for many hours, and in some cases, longer than a typical business day of eight hours. Such delays can cause added expense, preclude use in emergency situations, and reduce data quality due to degradation of reagents and increases in non-specific (background) signal during the protracted incubation times. Other limitations of conventional approaches include the need for analyte labeling steps and stringent washing steps, which introduce additional costs in terms of labor and equipment, and may impact data quality. Further, microplate based approaches use relatively large volumes of test samples and reagents.
One application for conventional arrays is in immunodiagnostics, such as to determine blood type. Blood typing entails determining the presence or absence of key surface antigens on human red blood cell (RBC) surfaces and/or determining the presence or absence of clinically relevant antibodies specific to RBC surface antigens. Conventional assays perform blood typing by detecting the agglutination of red blood cells (RBCs) upon the addition of the corresponding antibodies (see, for example, U.S. Pat. No. 4,894,347 to Hillyard et al.). Agglutination of a blood sample indicates a positive result for the antigen tested. Thus, blood typing methods are conducted in bulk to determine agglutination/adhesion, and indirectly measure the binding of the RBCs. Some methods for blood typing, referred to as “solid phase” methods, involve the immobilization of certain proteins or cells expressing certain proteins on a surface. These methods are generally done in bulk format. For example, a camera may detect whether most RBCs are on the walls of a well or at the bottom of a well by taking a single low-resolution image to see if the red color is at the central bottom portion of the well or along the edges. For strong signals and systems which are close to equilibrium, most cells may be found in similar states (most at the bottom, or most on the walls) giving rise to a reliable result for sufficient signal strength and incubation time.
Ensuring that blood typing is performed accurately is extremely important because RBCs from different individuals may have different antigens on their surfaces, and transfusion of whole blood or certain blood components from a donor having certain RBC antigens to a patient that lacks those antigens may cause an adverse transfusion reaction in the patient. This generally occurs if the patient has a natural immunization to the given antigens, which may occur with a non-matched ABO blood group, or if the patient has been immunized to blood antigens by prior exposure (e.g., Kell or Duffy group antigens). Thus, the potential for spurious results can occur due to possible immunization against blood cell antigens that is undetectable due to limits on measurement sensitivity or diminishing antibody concentration (titer) within an immunized patient's blood over time, or may also occur due to a possible variation in antibody titer and reactivity from person to person, or a potential variability in reagent specificity.
Present techniques for determining blood type are limited in their sensitivity, speed, and ability to test a sample for a large number of analytes. For example, the fact that conventional blood typing methods require agglutination or bulk surface binding, where most RBCs have to bind (and in many cases must bind strongly by forming multivalent attachments), limits the speed and sensitivity of the testing. Further, current technologies require a relatively large amount of blood (e.g., about 3 mL) for testing. The ability to type blood while utilizing a smaller volume would be of great utility, especially when dealing with newborns. The ability to reliably measure weaker binding levels, which have fewer bound cells and fewer attachments for each bound cell, would enable increased sensitivity (i.e., testing for lower titers with higher confidence), testing at much shorter times, or both benefits together.
With increased sensitivity, patient and donor blood can be more confidently matched. With increased speed, patients who have an urgent need for blood can be safely transfused with matched blood much more quickly. With increased capability for testing a large number of analytes, test costs can be reduced, additional tests can be adopted, and problems with specificity can be mitigated by testing against multiple related antibodies or reagent cells from multiple individuals. Expanding the test panel to include more tests of antigen variants or antibodies to these variants, can be valuable since variability is quite common due to genetic mutations and other variants, which is seen frequently in comparing populations with different ethnic backgrounds (and thus, genetic make-up), for example. If these advances can be made, the benefits could be extended to numerous other applications other than blood typing, including surface antigen characterization of stem cells, platelets, and cancer cells.